The present invention relates to a process for forming single-stranded circular DNA from single-stranded hairpin segments containing complementary sequences directing spontaneous circularization of said segments with subsequent ligation to form single circles.
Over the past several decades, research into molecular biology has been greatly enhanced by the plethora of new methodologies made available to workers in the field. Such methods have essentially helped to create the newer area of biotechnology and facilitated its emergence onto the commercial playing field. Especially advantageous has been the methods developed for replicating and amplifying short segments of specific DNA sequences so as to facilitate both the preparation of such sequences as well as amplification for the purpose of identifying desired sequences of DNA within a larger sample.
Most important in this regard has been the development of the polymerase chain reaction (PCR) as a means of exponentially amplifying desired segments of DNA using short primers that flank larger segments whose precise nucleotide sequence is not required to be known. Despite the enormous advantages conferred by such technology, there are certain drawbacks. These include the fact that PCR generates linear duplex sequences of DNA which require denaturation to be converted into single strand as well as further processing if circles are desired. In addition, the various steps of heating and cooling and the different enzymes required for the process make it expensive, time consuming and cumbersome. To respond to these problems, other methodologies have been devised, many of which are essentially modifications of the basic PCR process.
However, PCR remains inadequate for the rapid and inexpensive production of single-stranded circular DNAs. Such DNAs have found use as probes for various so-called target sequences existing within larger segments of single-stranded DNA. Often, such circles have been synthesized with discrete probe segments complementary to the sequences of the target DNA. In such cases, the circle is first synthesized as a so-called xe2x80x9copen circlexe2x80x9d and permitted to anneal to complementary sequences on a target single-stranded DNA. Once hybridized, the open circle is ligated to form a so-called padlock that is then used as a means of detecting the target DNA. [See: Nilsson et al., Padlock Probes: Circularizing Oligonucleotides for Localized DNA Detection, Science, 265, 2085-2088 (1994)]
Single-stranded circular DNA has been found useful in many different areas of biotechnology, both of an experimental as well as commercial nature. One important such use is as a substrate for rolling circle DNA replication. In this procedure, a single-stranded circle of DNA is mixed with a short strand of single-stranded complementary primer DNA and the two separate strands are allowed to anneal. After addition of a DNA polymerase, such as the Klenow fragment, the intact circle is used as a template by the enzyme and then replicated from the 3xe2x80x2-end of the primer strand. After the enzyme has gone around the circular template, it encounters the 5xe2x80x2-end of the primer, which is then displaced from the template strand so that the enzyme continues to move around the circular template while a long, unbroken single strand of DNA is generated. Such single strand has been referred to as single-stranded concatenated DNA (Ruth and Driver, WO 92/01813). The single-stranded circular products of the present invention are ideally suited for use as a substrate in such processes. The product prepared by the method according to the present invention can ultimately yield single-strand concatenated DNA having numerous different sequential segments that can act as probes, detection sites or restriction sites for further processing.
Heretofore, the products of so-called xe2x80x9crolling circle amplification,xe2x80x9d or RCA, have been used as binding sequences for probes containing complementary sequences for specific sequences located in target DNA whose presence it was desired to detect. In essence, the result is to amplify sequences contained in the circular template to facilitate detection of sequences contained in a target.
In addition, RCA has been used (see Ruth and Driver, WO 92/01813) to produce concatenated single-stranded DNA containing repeated sequences complementary to those contained in the single-stranded circular substrates but containing restriction sites. Thus, when the concatenated DNA is treated with restriction enzymes, it is cut into short, repetitive segments which can, if desired, be ligated to form structures complementary to the original circles. With the addition of primers and DNA polymerase, such process can be repeated to form copies of the original circles. The present invention eliminates the need for such multistep processes by duplicating the desired circles at the outset, thus eliminating the need for a second round of RCA.
Another useful aspect of RCA technology has been to generate circles of different size, combine them, and use these as probes for target DNAs having different target sequences. This has been done either by generating circles of different size and mixing them, or else by running multiple RCA reactions simultaneously in the same reaction mixture, with the relative amounts of starting material determining the relative amounts of products. However, one must first generate the different sized circles to use as substrates.
The methods according to the present invention eliminate the need for such cumbersome processes by providing an easy and effective means of generating circles of any desired sizes and sequences. Thus, using the methods disclosed herein, it is a simple matter to generate circles all of which have the same size, but whose nucleotide sequences are different, or to generate circles of different size and sequence, or any other conceivable combination, all at the same time. The same DNA polymerase will replicate all of the sequences together and their relative abundance in the product will be a function of their relative abundance in the starting mixture.
One of the problems of current RCA technology is that most starting circles are synthesized chemically (to facilitate predetermination of the nucleotide sequence or sequences contained within the circles). Such synthesis has made production of circles larger than about 100 bases both costly and time consuming. Of course, circles larger than about 200 nucleotides cannot be effectively prepared using current technology. Conversely, plasmid technology has not been of much use in this area because the needed starting circles must be single-stranded whereas plasmids are normally duplex DNAs.
Once stretches of single-stranded DNA have been synthesized, the circles are formed enzymatically through the use of ligase enzymes and employing a short guide oligonucleotide. Any remaining unreacted linear oligonucleotide and guide oligonucleotide are then digested with exonucleases. The entire process involves several steps, all of which work with varying efficiencies. The yield of circles by such processes is commonly less than 50% and other, undesired, forms are always present. A simpler and more efficient process for generating such circles is therefore desirable.
The methods according to the present invention involve a different approach from those described hereinabove. In accordance with the present invention, separate segments of single-stranded DNA, each possessing a first short sequence complementary to a second short sequence within the same oligonucleotide, are allowed to self-anneal, thereby forming a pseudocircular, or hairpin, oligonucleotide having an overhang composed of a portion of a terminal sequence of said single-stranded DNA.
It is a further object of the present invention to provide separate pseudocircular (i.e., hairpin) single stranded DNA structures that can readily be ligated to each other to form, after heat denaturation, enlarged single-stranded circular DNAs containing predetermined nucleotide sequences formed from the sequences of the starting segments.
It is another object of the present invention to provide a method of synthesizing single-stranded circular DNA molecules of varying sizes and sequences using pseudocircular DNA segments as a starting material and without the need for multiple cycles of synthesis and ligation.
It is also an object of the present invention to produce single-stranded circular DNA having predetermined sequences that can be readily replicated without the need for any linear intermediates.
It is likewise an object of the present invention to provide a means of producing multiple copies of single-stranded DNA circles of varying sizes and of any desired sequences for subsequent mixing to yield a single reaction mixture for further rolling circle replication.
It is another object of the present invention to provide a means of synthesizing single-stranded DNA circles capable of ready use in subsequent processes, such as rolling circle amplification.
It is a still further object of the present invention to provide hairpin oligonucleotides with complementary ends (i.e., complementary overhang regions) that comprise restriction sites for endonucleases to provide, after appropriate ligating and denaturation, single stranded circles whose RCA products can be cut by the same restriction enzyme to regenerate hairpin oligonucleotides, thereby providing, inter alia, an enzymatic source of oligonucleotides for production of circles without the need of chemical synthesis.
It is also an object of the present invention to provide hairpin oligonucleotides with coherent ends for ligation to DNA restriction fragments of any desired size and sequence to form circles for amplification by RCA.